Hi Emir:
Careful attention to detail goes a long way to ensure success.
1. Clean all your plates/gel boxes and prepare your solutions in a manner
that eliminates keratins from your hands, arms, and head from falling into
your experiment. Keratins are notorious for mucking things up --
especially if your protein of interest is about 70-90 kDa.
2. Deionize your acrylamide/bis with a mixed bed resin for an hour and
filter. Store in dark bottle.
3. After your gel has been poured and polymerized, wrap it up in wet
paper towels soaked in running buffer. then wrap in saran wrap and store
overnight to allow the free radicals from persulfite to dissipate. This
will address your unpoly acry concern.
4. Sample preparation is crucial. Don't use urea and boil -- you will
carbamylate your N-term. Just use freshly made 2% SDS, 0.125 M Tris-Hcl,
pH 6.8, 5% glycerol, and a touch of dye -- heat to 37C for 15 min.
This assumes your sample will go into solution. Each sample is different.
You may have to use more SDS and higher temps. Spin out insoluble debris.
5. The type of PDVF is important. Millipore makes different kinds for
differnnt proteins -- because at the standard 0.45 um porosity, protiens
will go right through the mb. Even at 0.2 um. Try to buy 0.1 um -- and
then even use 3 sheets of PVDF. Proteins blow right through these mbs.
Remember to soak in MeOh first, then equilibrate in run bf.
~~
6. I like to use 10 mM CAPS pH 11.0 with 20% methanol as a running
buffer. You will need abou 0.5 Amps of current over 30-40 min -- start
with chilled buff and use an ice dam. Your run buffer will heat up alot.
If you feel that the pH is too high, then you can use regular ol' running
buffer (tris-glycine). CAPS < 11.0 won't work cause it won't conduct
electricity. If you use tris-glycine, you must wash wash wash (below)
7. After run, stain and destain. In my experience, I've never had a
problem here -- but watch out for ppt in your coomassie.
8. Begin the wshing -- use the best water you can afford. Use an ultra
clean glass casserole dish -- use lots of water and change often.
you can wash over the weekend (convenient). for my last wash, i use
HPLC water. For mini gels, I use 1-2 liters per rinse.
This step cannot be emphasized enough. when in doubt, wash more.
9. Let your sequence guy cut the band out.
Hope this helps,
James A. Bassuk, Ph.D.
Research Scientist
Division of Pediatric Urology
Children's Hospital & Regional Medical Center
4800 Sand Point Way N.E.
P.O. Box 5371, MailStop CH-78
Seattle, WA U.S.A. 98105-0371
office: 206-528-2623
fax: 206-528-2617
email: jbassu at chmc.org
Assistant Professor
Department of Urology
University of Washington School of Medicine
1959 N.E. Pacific Avenue
Box 356510
Seattle, WA 98195
office: 206-685-3780
lab: 206-685-3080
fax: 206-543-3272
email: bassuk at u.washington.edu
On Fri, 2 Nov 2001, Emir Khatipov wrote:
> Hello All,
> Could someone recommend me a good protocol for SDS-PAGE and subsequent
> transfer of proteins onto PVDF membrane for subsequent Edman sequencing?
>> Which PVDF membrane did you use, how did you deal with non-polymerized
> acrylamide that can modify proteins and impair sequencing, what transfer
> buffers worked for you best, etc. Should I use PAGE buffers different from
> Tris-glycine (Borate?), and do I have to care about glycine on Page stage at
> all? I know that CAPS buffer is a good alternative for Tris glycine transfer
> buffer, but I am concerned that high pH of the buffer (pH 11) will cause
> modification of side chains of amino acids. In other words, I good detailed
> working protocol would be very much appreciated, or pointers to published
> resources.
>> Thank you.
> Emir
>> University of Chicago
> Radiation Oncology
>>>